This research will develop, characterize and optimize a novel synthetic gene carrier for the delivery of the human cystic fibrosis transmembrane conductance regulator encoding DNA to human airway epithelial and submucosal cells in the lung.The delivery system consists of a targeting ligand, membrane destabilizing component, nuclear entry component, DNA condensor and DNA masking component that form a complex directly with the DNA. The targeting ligand is attached to the DNA via a DNA intercalator or via electrostatic interactions. The membrane destabilizing component is a membrane active peptide that facilitates transmembrane transfer of DNA. The current version of the complex is between 20 to 100 times more effective than the cationic lipid DOTMA at mediating transfection of cells in culture. The research plan has four interacting components: l. Synthesis of the delivery ligands and membrane destabilizers 2. Optimization and characterization of the transfection system in cultured cells and rat lung using reporter genes, 3. Transfection of CFTR into cultured human primary airway and serous cells and assessment of function. 4. Transfection of CFTR into rat and other animal model lungs via the airway or the parenteral route and characterization of the extent, duration and location of expression of human CFTR. The synthesis and detailed physico-chemical characterization of the delivery complex will be emphasized in order to correlate the various components with successful transfection. Various ligands will be attached to the complex to exploit receptor mediated endocytosis to accelerate entry of the DNA complex into airway epithelial and submucosal cells. Nuclear localization peptide-acridine conjugates will be attached to DNA to increase nuclear uptake. To increase Serous cell specific expression, cell specific promoters and cell surface targeting ligands will be included in the complex. The level, duration and percentage of cells expressing transfected human CFTR gene will be estimated by Northern blots and antibody staining. More importantly the function of the transfected CFTR will be quantitated in human Hela and HL60 cells in culture using chloride sensitive fluorescent indicators. Function will also be measured in human CF primary cultures of epithelial and tracheobroncheal glands on supported monolayers using Ussing chambers. To determine whether methodology that works in vitro can function in vivo we will transfect reporter genes and human CFTR into the lungs of rats via the airways and by the parenteral route. For parenteral DNA delivery, agents that mask the DNA, to decrease clearance by the reticuloendothelial cells, will be incorporated into the complex. In addition, strategies that transiently open the venules in the upper airways will be used to increase extravasation of the complex to the submucosal space. Our ultimate goal is to develop a non-viral gene therapy treatment for Cystic Fibrosis.